1. Field of Invention
The invention relates to a fluorescent polymer fine particle, a method for forming thereof, a fluorescence detection kit, and a method for detecting fluorescence. Specifically, the invention relates to a fluorescent polymer fine particle, a method for forming thereof, a fluorescence detection kit comprising a fluorescent polymer fine particle, and a method for detecting fluorescence comprising using a fluorescent polymer fine particle.
2. Description of the Related Art
In order to visualize or quantify a substance of a minute amount, various labeling substances have been developed. In fields requiring a particularly high sensitivity, radioisotopes are representative labeling substances, and tritium and radioactive iodine have been utilized as representative examples. However, as the radioactive substances involve various difficulties in disposal after use and in handling, methods alternative to the radioactive substances have been developed. Such methods include, for example, an enzyme labeling method (utilizing peroxidase, alkaline phosphatase, glucose oxidase or β-D-galactosidase), and a fluorescent labeling method (utilizing fluorescein or rhodamine).
However, these methods involve a drawback of being deficient in the absolute sensitivity as a label.
In order to improve the precision and sensitivity of measurement, a time-resolved fluorescence measurement has been developed (cf. JP-A-61-128168) as an extension of the fluorescent labeling method. This method is based on irradiating a fluorescent substance of a long fluorescence extinction time, as represented by an europium chelate, with a pulsed excitation light, and measuring the fluorescence after a certain time namely after the termination of the direct excitation light and the extinction of fluorescence of short duration resulting from ambient substances, thereby measuring a fluorescence specific to europium.
It is also attempted, in order to further improve the sensitivity, to enclose such europium chelate or a dye in polystyrene particles, then to coat the surface of the polystyrene particles with an antigen or an antibody to prepare a reagent, and to visually detect the polystyrene particles immobilized by the antigen-antibody reaction (for example cf. JP-A-2000-345052).
However, such known method of preparing a labeling substance by enclosing a dye or a fluorescent substance in polystyrene particles, though being capable of attaining a certain sensitivity by simple operations, is insufficient in sensitivity, and a further improvement in the sensitivity has been desired.
Also because of the fact that the particle surface is constituted of hydrophobic polystyrene, the method has been utilized with various modifications such as, (1) after bonding a functional molecule such as an antigen or an antibody desired for coating, coating the unbonded surface with a protein or various biosubstance-like materials thereby masking the hydrophobicity of polystyrene, or (2) adding a surfactant in the liquid phase at the reaction thereby preventing mutual interaction of the polystyrene particles. Nevertheless, errors in judgment may result from a non-specific reaction.
On the other hand, in order to suppress the non-specific reaction resulting from the polystyrene particles, there have been developed a technology of preparing fine particles of a core-shell configuration formed by a core of a water-insoluble polymer compound and a hydrophilic shell having a reactive group, and enclosing a fluorescent dye in the core portion, and a composition for fluorescence analysis, capable of stably maintaining thus obtained high fluorescence intensity (for example cf. WO 2002/097436 pamphlet and JP-A-2005-49207).
However, the method utilizing such fine particles of core-shell configuration may suppress noises induced by the non-specific reactions, it is unsatisfactory in the fluorescence intensity. Further, such fine particles are insufficient in the stability in time, and may lose the fluorescence intensity within the period of use.
Also lanthanoid dyes have an excitation wavelength within the ultraviolet region, and most of the hitherto known ones have excitation maxima at wavelengths shorter than 350 nm. For this reason, the light source for excitation is inevitably restricted, and an expensive light source has to be used.